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WO1997000849A1 - Nouveaux composes de sulfure de vinyle et leur procede de preparation - Google Patents

Nouveaux composes de sulfure de vinyle et leur procede de preparation Download PDF

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Publication number
WO1997000849A1
WO1997000849A1 PCT/US1996/009249 US9609249W WO9700849A1 WO 1997000849 A1 WO1997000849 A1 WO 1997000849A1 US 9609249 W US9609249 W US 9609249W WO 9700849 A1 WO9700849 A1 WO 9700849A1
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Prior art keywords
compound
dioxetane
group
formula
optionally
Prior art date
Application number
PCT/US1996/009249
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English (en)
Inventor
Hashem Akhavan-Tafti
Zahra Arghavani
Robert A. Eickholt
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Lumigen, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Lumigen, Inc. filed Critical Lumigen, Inc.
Priority to DE69624374T priority Critical patent/DE69624374T2/de
Priority to AT96923225T priority patent/ATE226188T1/de
Priority to CA002222788A priority patent/CA2222788C/fr
Priority to EP96923225A priority patent/EP0869938B1/fr
Priority to AU63793/96A priority patent/AU709184C/en
Priority to US08/981,448 priority patent/US5883287A/en
Priority to JP9503860A priority patent/JP3023993B2/ja
Publication of WO1997000849A1 publication Critical patent/WO1997000849A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/11—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/16—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/56—Ring systems containing bridged rings
    • C07C2603/58—Ring systems containing bridged rings containing three rings
    • C07C2603/70—Ring systems containing bridged rings containing three rings containing only six-membered rings
    • C07C2603/74—Adamantanes

Definitions

  • the present invention relates to a novel process for the preparation of stable chemiluminescent 1,2-dioxetane compounds which can be triggered to generate light.
  • Stable, triggerable dioxetanes prepared by the present process are preferably of the formula:
  • the present invention also relates to novel sulfur- substituted alkenes (vinyl sulfides) preferably of the formula:
  • R 2 R 3 ox and stable triggerable sulfur-substituted 1,2-dioxetanes preferably of the formula:
  • the enzymatically triggerable dioxetanes are now undergoing widespread use as substrates for marker enzymes in numerous applications including immunoassays, gene expression studies, Western blotting, Southern blotting, DNA sequencing and the identification of nucleic acid segments in infectious agents.
  • New processes for the preparation of existing and new triggerable dioxetanes are desirable to advance the state of the art. Processes which permit the preparation of dioxetane compounds which are difficult or impossible to prepare by known methods would be particularly desirable. The process of the present invention provides such means.
  • Dioxetane compounds prepared by the process of the present invention can be used in assay methods to signal the presence or amount of an analyte, in emergency and novelty lighting applications and as light standards for luminometer calibration.
  • Dioxetane compounds which are triggered by an activating agent to produce light are useful in immunoassays and the detection of nucleic acids, antibodies, haptens and antigens by generally known methods.
  • the present invention relates to a general process for the preparation of stable aryl group-substituted 1,2-dioxetanes (III) further substituted on the dioxetane ring with an alkoxy, aryloxy or acyloxy group which dioxetane can be triggered to generate chemiluminescence.
  • the process involves the substitution of an alkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy or acyloxy group OR 5 for a thioalkyl or thioaryl group SR 4 on the dioxetane ring of a sulfur-substituted dioxetane (II) mediated by an electrophilic compound E-Y.
  • Sulfur-substituted dioxetanes of the present invention are prepared by addition of oxygen to a vinyl sulfide (I) .
  • the sequence of reactions is shown in the Scheme below.
  • the present invention represents the first demonstration of the synthesis of a dioxetane by replacing one of the dioxetane ring substituents of a different precursor dioxetane.
  • the ability to selectively replace one of the ring substituents readily allows the synthesis of a variety of new dioxetane compounds from a common intermediate.
  • Sulfur-substituted dioxetanes useful in practicing the process of the present invention can be of the formula:
  • R x and R 2 are organic groups providing sufficient stability to the dioxetane to permit conversion to an alkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy or acyloxy-substituted dioxetane.
  • the groups R ⁇ and R 2 are chosen from branched chain or cyclic alkyl, substituted alkyl or heteroalkyl groups.
  • R x and R 2 can optionally be joined together to form a cyclic or polycyclic group which is spiro-f sed to the dioxetane ring.
  • the group R 4 is selected from (C 1 -C 20 ) alkyl, (C 7 -C 30 ) aralkyl and (C 6 -C 30 ) aryl groups which can optionally contain non-interfering substituents and optionally contain N, 0, S, P or halogen heteroatoms within the alkyl, aralkyl or aryl group.
  • the group R 3 is selected from aryl, biaryl, heteroaryl, fused ring polycyclic aryl or heteroaryl groups in which one or more of the ring hydrogens can be replaced by an atom or group selected from halogens, alkyl, alkoxy, substituted alkoxy, carbonyl, carboxyl, amino and alkylamino groups.
  • the X group can be any protecting group which serves to block formation of the aryloxide anion and which can be replaced or removed as desired by an activating agent to form the aryloxide anion.
  • Representative OX groups include hydroxyl, alkoxy, substituted alkoxy (e.g.
  • acyloxy having the formula OOCR 10 wherein R 10 is selected from alkyl and aryl groups containing 2 to 20 carbon atoms, trialkylsilyloxy, triarylsilyloxy, aryldialkylsilyloxy, OPO(OR 8 ) 2 wherein R 8 is an organic group, oxygen pyranoside including, without limitation, ⁇ -D-galactosyloxy and ⁇ -D-glucuronidyloxy groups.
  • the process is used to form a sulfur-substituted dioxetane (V) wherein the R. and R 2 groups are joined together as a polycyclic alkyl group spiro-fused to the dioxetane ring as represented by the formula:
  • L C is the polycyclic alkyl group. Most preferred is a sulfur-substituted dioxetane wherein the R 6 C group is an adamantyl group with optional non-hydrogen substituents.
  • Electrophilic agents useful in practicing the present invention include but are not limited to halogens including Cl 2 , Br 2 , l 2 , ICI and IBr, hydrogen peroxide, singlet oxygen, pseudo-halogens such as N-chlorosuccinimide (NCS) , N-bromosuccinimide (NBS) and N-iodosuccinimide (NIS) , alkylating agents including alkyl halides and alkyl sulfates and sulfonates, transition metal salts, especially salts of mercury, silver and gold, and Lewis acids such as titanium tetrachloride.
  • halogens including Cl 2 , Br 2 , l 2 , ICI and IBr
  • pseudo-halogens such as N-chlorosuccinimide (NCS) , N-bromosuccinimide (NBS) and N-iodosuccinimide (NIS)
  • alkylating agents including alkyl halides
  • the compound of structure E-Y may serve as both the electrophilic agent and the hydroxylic compound R 5 0"M + .
  • Compounds of this type described generically as (R 5 0 " ) n M +n wherein M is a metal with a strong propensity to react with sulfur, such as silver, gold and especially, mercury, wherein n is 1,2 or 3 and wherein R 5 0 ⁇ is an anion of a hydroxylic compound, in particular a carboxylate anion are effective to replace the SR 4 group of a sulfur-substituted dioxetane with an OR 5 group.
  • a preferred compound of this type is mercuric acetate Hg(OAc) 2 .
  • Photosensitizers useful in practicing the process of the invention include compounds known in the art to produce singlet oxygen upon irradiation with visible light in the presence of ground state oxygen.
  • Exemplary photosensitizers include methylene blue, Rose Bengal, eosin, erythrosin, rhodamines, porphyrins, metal porphyrins and fullerenes.
  • Photosensitizers may be employed as the soluble dye or linked to an insoluble support such as silica or a polymer bead.
  • Preferred photosensitizers are methylene blue or polymer-bound Rose Bengal.
  • a vinyl sulfide containing a sulfur substituent SR 4 wherein R 4 is an organic group containing 1 to 20 carbon atoms and optionally heteroatoms is converted by low temperature photooxygenation to the corresponding sulfur- substituted 1,2-dioxetane by addition of singlet oxygen to the double bond.
  • Progress of this reaction is readily monitored by thin layer chromatography (TLC) or 1 H NMR by observing the disappearance of the vinyl sulfide. Additionally, heating a small portion of the reaction solution leads to easily detectable chemiluminescence indicating formation of the sulfur-substituted dioxetane.
  • Sulfur-substituted dioxetane compounds of relatively lower thermal stability are preferably not isolated at this point but instead directly reacted at a low temperature with a compound containing a hydroxyl group or a salt thereof.
  • Sulfur-substituted dioxetane compounds of relatively higher thermal stability can be first isolated before reaction with a compound containing a hydroxyl group or a salt thereof.
  • An electrophilic compound is added at low temperature to the sulfur-substituted dioxetane in an amount ranging from about 0.5 mol to about 1.5 mol of electrophilic compound per mol of dioxetane based on complete conversion of the vinyl sulfide. It is especially preferred that the photooxygenation and addition of electrophilic compound be performed at or below about -40 °C; use of a Dry Ice-isopropyl alcohol mixture, ca. -78 °C, for cooling is particularly suited for this purpose.
  • the hydroxylic compound is permitted to react with the dioxetane to effect replacement of the SR 4 group with the OR 5 group.
  • Vinyl sulfide compounds (I) useful in practicing the present invention are preferably of the formula:
  • R and R 2 are organic groups which can optionally be joined together to form cyclic or polycyclic groups, wherein R 4 is selected from (C ⁇ C- j o) alkyl, (C 7 -C 30 ) aralkyl and (C 6 -C 30 ) aryl groups and optionally containing heteroatoms, wherein R 3 is selected from aryl, biaryl, heteroaryl, fused ring polycyclic aryl or heteroaryl groups which can optionally contain non-interfering substituents and wherein X is a protecting group.
  • R. and R 2 groups are selected from branched chain alkyl, cycloalkyl and aryl groups containing 3 to 20 carbon atoms and optionally heteroatoms.
  • a vinyl sulfide having the formula:
  • R 3 is an optionally substituted phenyl, naphthyl or other fused-ring aryl group. It is especially preferred that R 3 is a phenyl group in which an OX group is oriented meta to the dioxetane ring group as shown below.
  • the phenyl ring can optionally contain additional ring substituents independently selected from halogens, alkyl, substituted alkyl, alkoxy, substituted alkoxy, carbonyl, carboxyl, amino and alkylamino groups.
  • C is more preferably a polycyclic group, preferably an adamantyl group optionally having one or more non-interfering substituent groups selected from halogens, alkyl, substituted alkyl, alkoxy, substituted alkoxy, carbonyl, carboxyl, phenyl, substituted phenyl, amino and alkylamino groups covalently bonded thereto.
  • Vinyl sulfides useful in practicing the process of the present invention can be prepared by art-known methods for the preparation of vinyl sulfides. An exemplary proces ⁇ for the preparation of vinyl sulfides is disclosed in T. Mukaiyama, K, Saigo, Chem. Lett., 479-82 (1973).
  • vinyl sulfide (IV) is formed by coupling a ketone compound and a thioester compound with a low valent titanium reagent exemplified by the following reaction: Scheme 3.
  • the low valent titanium reagent which is used in excess, is prepared by reacting a titanium salt, preferably TiCl 3 or TiCl 4 with a metallic reducing agent selected from lithium, sodium, potassium, zinc and zinc-copper alloys or a metal hydride including, without limitation, sodium hydride, potassium hydride, aluminum hydride, lithium aluminum hydride or potassium triethylborohydride in the presence or absence of an amine base in an aprotic solvent, preferably tetrahydrofuran.
  • the ratio of ketone to thioester is preferably from about 1:3 to about 3:1.
  • the reaction between the ketone and thioester is conducted between about 0 °C and the reflux temperature of the solvent, preferably between about 20 °C and about 70 °C
  • Thioalkyl- and thioaryl-substituted dioxetanes of the present invention are surprisingly stable and can be manipulated at temperatures of -10 to 25 °C
  • Reaction of the thioalkyl- and thioaryl-substituted dioxetanes of the present invention with an activating agent to cleave the O-X bond and remove the X group yields an unstable oxide intermediate dioxetane compound is formed which decomposes and releases electronic energy to form light and two carbonyl-containing compounds of the formula:
  • Stable alkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy or acyloxy-substituted dioxetanes which can be prepared by the process of the present invention are of the formula: wherein R x and R 2 are organic groups which provide stability and which can optionally be joined together to form a cyclic or polycyclic group which is spiro-fused to the dioxetane ring, wherein R 5 is selected independently from the group consisting of (C 1 -C 20 ) alkyl groups, (C 2 -C 20 ) alkenyl groups, (C 2 -C 20 ) alkynyl groups, (C 6 -C 30 ) aryl groups, (C 7 -C 30 ) aralkyl groups and (C 1 -C 20 ) acyl groups.
  • R 5 can optionally be substituted with one or more substituents including, without limitation, hydroxy, alkoxy, halogen, cyano, nitro, amino, imine, ketone, aldehyde, carboxylic acid, carboxylic ester, carboxamide, thiol, thioester, trialkyl ⁇ ilyloxy, triarylsilyloxy and alkyldiarylsilyloxy substituents.
  • the group R 3 is selected from aryl, biaryl, heteroaryl, fused ring polycyclic aryl and fused ring polycyclic heteroaryl groups which can optionally be substituted with non-interfering groups.
  • X is a group which can be removed by an activating agent as is generally known in the art to form an unstable oxide intermediate dioxetane compound.
  • X can be a group such as a hydrogen atom or a labile group which can be replaced with another removable group without triggering the decomposition of the dioxetane.
  • Representative OX groups include hydroxyl, alkoxy, substituted alkoxy (e.g.
  • acyloxy having the formula OOCR 10 wherein R 10 is selected from alkyl and aryl groups containing 2 to 20 carbon atoms, trialkylsilyloxy, triarylsilyloxy, aryldialkylsilyloxy, OPO(OR 8 ) 2 wherein R 8 is an organic group, OP0 3 2 ⁇ salts and oxygen pyranoside including, without limitation, ⁇ -D-galactosyloxy and ⁇ -D-glucuronidyloxy group ⁇ .
  • a preferred dioxetane compound which can be made by the process of the present invention is a stable dioxetane of the formula:
  • R 6 C is selected from cyclic and polycyclic organic groups, which can optionally be substituted with non ⁇ interfering groups and which is spiro-fused to the dioxetane ring and which provides thermal stability, wherein R 3 , R 5 and X are as defined above so that when the dioxetane is triggered to remove the X group by an activating agent an unstable oxide intermediate dioxetane compound is formed which decomposes and releases electronic energy to form light and two carbony1-containing compounds of the formula:
  • the group R ⁇ C is a polycyclic organic group spiro-fused to the dioxetane ring, containing 6 to 30 carbon atoms and which can optionally be substituted with non-interfering groups and which provides thermal stability.
  • the group R ⁇ C is more preferably an adamantyl group optionally having at least one substituent group selected from halogens, alkyl, substituted alkyl, alkoxy, substituted alkoxy, carbonyl, carboxyl, phenyl, substituted phenyl, amino and alkylamino groups covalently bonded thereto.
  • the group R 3 is a phenyl or naphthyl group.
  • R 3 is a phenyl group in which the OX group as defined above is oriented meta to the dioxetane ring group as shown below.
  • the phenyl ring can optionally contain additional ring sub ⁇ tituents independently selected from halogens, alkyl, substituted alkyl, alkoxy, ⁇ ub ⁇ tituted alkoxy, carbonyl, carboxyl, amino and alkylamino groups.
  • the stable 1,2-dioxetane compounds have long half-lives at room temperature, typically > 1 year, but can be triggered by an activating agent to decompose rapidly with half-lives ranging from seconds to a few minutes depending on the microenvironment where the dioxetane is located.
  • Dioxetanes wherein Ar is an optionally substituted (C 6 -C 30 ) aryl group and wherein X is a removable group are highly stable compounds which produce visible light when triggered to remove X.
  • the hydroxylic compound R 5 OH may itself be another dioxetane molecule with a phenolic group.
  • chain ⁇ of directly linked, oligomeric or polymeric dioxetane ⁇ are formed.
  • such oligomeric or polymeric dioxetanes can serve as the hydroxylic compound in order to build chain structures of greater length.
  • a requirement for this proce ⁇ s is that the OX group of the monomeric, oligomeric or polymeric dioxetane serving as the R' 5 OH component be present a ⁇ OH.
  • An example ⁇ howing formation of a dimeric bi ⁇ -dioxetane (VII) is illustrated below to aid in under ⁇ tanding the proce ⁇ s. Scheme 5.
  • the intermediate carbonium ion can serve as its own R 5 OH component; i.e. when X is H a self-condensation can occur to produce an oligomeric or polymeric dioxetane chain compound.
  • a sub ⁇ stoichiometric amount of a competing R' 5 OH compound is required to effect chain termination.
  • a po ⁇ ible dioxetane compound (VIII) prepared by thi ⁇ proce ⁇ i ⁇ illu ⁇ trated to aid in under ⁇ tanding the proce ⁇ .
  • the hydroxylic compound can be a carboxylic acid R 9 COOH, a ⁇ alt thereof or it can be a ⁇ the electrophilic compound in the manner described above.
  • Dioxetanes wherein R g is an optionally sub ⁇ tituted (C ⁇ C* ⁇ ) alkyl or aryl group and wherein X is a removable group are also stable compounds which emit light when triggered to remove X.
  • a significant advantage of the present proces ⁇ for preparing alkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy or acyloxy- ⁇ ub ⁇ tituted dioxetanes is that a single vinyl sulfide precursor compound can be converted into any of a large number of other dioxetane compounds limited only by the availability of the hydroxylic compound R 5 -OH or ⁇ alt R 5 0 _ M + . Since the OR 5 group i ⁇ not introduced until after formation of the dioxetane ring, it is possible to prepare dioxetane compounds which would be problematic to prepare by the prior art process of photooxygenating a vinyl ether precursor.
  • the present process is amenable to the preparation of dioxetanes with groups which could interfere with the photooxygenation either by quenching of singlet oxygen, or by quenching or bleaching the photosensitizer.
  • the present proces ⁇ i ⁇ al ⁇ o amenable to the preparation of dioxetanes with groups which make the vinyl ether more electron-deficient and therefore less reactive or unreactive to singlet oxygen.
  • the present process further permits the preparation of dioxetanes with groups such as C-C double bonds which are themselves reactive toward singlet oxygen.
  • Dioxetanes prepared by the proces ⁇ of the pre ⁇ ent invention can be u ⁇ ed in a process for generating light which comprises reacting an activating agent with a stable 1,2-dioxetane of the formula (III):
  • the activating agent can be a chemical, including an enzyme, which reacts catalytically or stoichiometrically to trigger the dioxetane.
  • exemplary activating agent ⁇ are disclosed, for example, in U.S. Patent 4,857,652 the relevant portion of the disclosure of which is incorporated herein by reference.
  • Activating agents which react catalytically or stoichiometrically to trigger the dioxetane by removal of the X group are well known in the art. Reactions can be conducted in aqueous solution, in which case it is often desirable to use one or more art-known chemiluminescence enhancers to increase or prolong light emission. Reactions can also be conducted in polar, aprotic solvent ⁇ ⁇ uch as dimethyl sulfoxide, N,N- dimethylformamide or acetonitrile.
  • the proces ⁇ for generating light can be performed in ⁇ olution or on the ⁇ urface of a solid support such as a membrane.
  • Dioxetanes prepared by the proces ⁇ of the present invention can further be incorporated into a chemilumin ⁇ escent composition which comprises a dioxetane, one or more enhancer substances and optional fluorescers.
  • Enhancers are sub ⁇ tance ⁇ which increase the amount of light produced on triggering the dioxetane.
  • Enhancer ⁇ ubstances well known in the art increase chemiluminescence either by providing a hydrophobic environment in which the light emitting reaction can occur or through energy transfer to a fluorescent compound held in proximity to the dioxetane.
  • the ethyl acetate layer was then dried over magnesium sulfate, filtered and concentrated in vacuo yielding a viscous yellow oil which smelled of thiol.
  • the oil was chromatographed on silica gel using methylene chloride as eluent. The de ⁇ ired vinyl ⁇ ulfide and the starting ketone were recovered. The solvents were removed in vacuo from the first eluted fraction yielding 8.57 g of light yellow oil which gradually solidified over the course of several days.
  • the ethyl acetate layer was then dried over magnesium ⁇ ulfate, filtered and concentrated in vacuo yielding a viscous yellow oil which smelled of the thiophenol.
  • the oil was chromatographed on silica gel using methylene chloride as eluent.
  • the vinyl sulfide was collected in two fractions, the first being contaminated with the thiophenol, the second being pure product.
  • the first fraction was chromatographed on silica gel using 10-20% ethyl acetate in hexane as eluent yielding a second crop of vinyl sulfide which combined with the previous crop yielded
  • a small photooxygenation apparatus wa ⁇ charged with 54 mg of the vinyl ⁇ ulfide, 1-2 mg of methylene blue, 5 mL of CH 2 C1 2 (dried over MgS ⁇ 4) and 5 mL of CF 3 CH 2 OH. The resulting solution was then cooled to -40 °C with oxygen bubbling through it. After several minute ⁇ , the reaction solution was irradiated with a 1000 W sodium lamp for 24 min to produce the dioxetane as shown by the appearance of a new material by TLC which eluted immediately below the alkene and emitted light when heated.
  • Example 5 Synthesis of Compound 5. 4- (2,2,2-trifluoro- ethylthio) -4- (3-hydroxyphenyl)spiro[1,2-dioxetane-3,2 ' - tricyclo[3.3.1.1 3 - 7 ] decane].
  • reaction solution was irradiated with a 1000 W sodium lamp for 45 min to produce mainly the dioxetane as shown by the light emitted on heating the TLC plate.
  • the solution was concentrated and the product isolated by preparative TLC using 20% ethyl acetate in hexane yielding 30.4 mg of the product as a ⁇ lightly yellow oil.
  • Example 6 Synthesis of Compound 6. 4- (3-Hydroxyphenyl) -4- methoxy ⁇ piro[1,2-dioxetane-3 ,2 ' -tricyclo[3.3.1.1 3 - 7 ] decane] .
  • the vinyl ⁇ ulfide of Example Kb) (0.113 g) wa ⁇ irradiated for 15 min in 20 mL of methanol containing a few crystals ( ⁇ 1 mg) of methylene blue with continuous oxygen bubbling at -78 °C using a 1000 W Na lamp. Iodine (0.096 g) and 1 mL of 30% H 2 0 2 were added and the mixture warmed to room temperature.
  • Example 7 Alternate Synthesis of Compound 6.
  • the vinyl sulfide of Example Kb) (0.104 g) was irradiated for 23 min in 10 mL of CH 2 C1 2 containing a few crystal ⁇ ( ⁇ 1 mg) of methylene blue with continuou ⁇ oxygen bubbling at -78 °C u ⁇ ing a 1000 W Na lamp.
  • 28 ⁇ L of methanol (2 eq. ) was added and the solution warmed to room temperature.
  • TLC showed a new material which emitted blue light when the plate wa ⁇ heated.
  • the vinyl sulfide of Example Kb) (0.131 g) was irradiated for 13 min in 20 mL of 2-propanol containing a few crystal ⁇ ( ⁇ 1 mg) of methylene blue with continuous oxygen bubbling at -78 °C using a 1000 W Na lamp.
  • Iodine (0.111 g) and 1 mL of 30% H 2 0 2 were added and the mixture warmed to room temperature.
  • Progress of the reaction was monitored by the appearance of a new material by TLC (40% ethyl acetate in hexane) which emitted blue light when the plate was heated and by observing the color of chemiluminescence from an aliquot of the reaction solution reacted with O.l M TBAF in DMSO.
  • the yellow emi ⁇ sion of dioxetane 1 was gradually replaced by the blue emi ⁇ ion of dioxetane 7 over a period of ⁇ everal hours.
  • the solution was concentrated to 1-2 mL and separated on preparative TLC with 10% ethyl acetate in hexane.
  • Example 9 Synthesis of Compound 8. 4- (3-Hydroxyphenyl) -4- (2-propenyloxy)spiro[l,2-dioxetane-3,2 ' -tricyclo[3.3.l.l 3 - 7 ] decane].
  • the vinyl sulfide of Example Kb) (0.100 g) was irradiated in 20 mL of CH 2 C1 2 containing a few crystal ⁇ ( ⁇ 1 mg) of methylene blue with continuous oxygen bubbling at -78 °C using a 1000 W Na lamp.
  • Example 10 Synthesis of Compound 9. 4- (2-Cyanoethoxy) -4- (3-hydroxyphenyl) ⁇ piro [1,2-dioxetane-3,2 ' -tricyclo- [3.3.l.l 3 - 7 ]decane] .
  • Example 11 Synthesis of Compound 10. 4- (2-Hydroxyethoxy) - 4- (3-hydroxyphenyl) spiro[1, 2-dioxetane-3 , 2 ' -tricyclo- [3.3.l.l 3 - 7 ]decane] .
  • the vinyl sulfide of Example Kb) (0.099 g) was irradiated for 11.5 min in 15 mL of CH 2 C1 2 containing a few crystals ( ⁇ 1 mg) of methylene blue with continuous oxygen bubbling at -78 °C using a 1000 W Na lamp. TLC indicated that the vinyl sulfide was consumed.
  • N- Chlorosuccinimide (0.044 g) was added and the mixture maintained at -78 °C for 15 minutes.
  • Ethylene glycol (37 uL) was added and the solution warmed to room temperature.
  • Progress of the reaction was monitored by the appearance of a new material by TLC (20% ethyl acetate in hexane) which emitted blue light when the plate was heated and by observing the color of chemiluminescence from an aliquot of the reaction ⁇ olution reacted with O.l M TBAF in DMSO.
  • the yellow emission of dioxetane 1 was gradually replaced by the blue emission of dioxetane 10 over a period of an hour.
  • Example 12 Synthesis of Compound 11. 4-(3-Hydroxyphenyl) - 4-phenoxyspiro[l,2-dioxetane-3,2 ' -tricyclo[3.3. l.l 3 - 7 ] decane] .
  • the vinyl sulfide of Example Kb) (0.106 g) was irradiated for 10 min in 20 mL of CH 2 C1 2 containing a few cry ⁇ tals ( ⁇ 1 mg) of methylene blue with continuous oxygen bubbling at -78 °C using a 1000 W Na lamp. TLC indicated that the vinyl sulfide was consumed.
  • N-Chlorosuccinimide (0.047 g) was added and the mixture maintained at -78 °C for 15 minutes. Phenol (83 mg) was added and the solution warmed to room temperature. Progre ⁇ of the reaction wa ⁇ monitored by the appearance of a new material by TLC (20% ethyl acetate in hexane) which emitted blue light when the plate wa ⁇ heated and by ob ⁇ erving the color of chemiluminescence from an aliquot of the reaction solution reacted with O.l M TBAF in DMSO. The yellow emission of dioxetane 1 was gradually replaced by the blue emission of dioxetane 11 over a period of an hour.
  • the solution was concentrated and the re ⁇ idue chromatographed on a ⁇ ilica prep. TLC plate with 20% ethyl acetate in hexane.
  • the major band was i ⁇ olated and found to contain a mixture of phenol, ca. 60 mg of dioxetane 11 and ca. 5 mg of adamantanone.
  • Example 13 Synthesis of Compound 12. 4-Acetoxy-4- (3- hydroxyphenyl)spiro[1, 2-dioxetane-3 ,2 ' -tricyclo [3.3.l.l 3 ' 7 ] decane].
  • the vinyl sulfide of Example Kb) (50 mg) was irradiated for 25 min in 20 mL of CH 2 C1 2 containing 2 mg of methylene blue with continuous oxygen bubbling at -78 °C using a 1000 W Na lamp.
  • Mercuric acetate 400 mg
  • TLC indicated the formation of two new products which emitted light upon heating the plate.
  • Example 14 Synthesis of Compound 13.
  • a ⁇ mall photooxygen ⁇ ation apparatus was charged with 99.1 mg of the vinyl sulfide of Example 3(a), 1-2 mg of methylene blue, 10 mL of CH 2 C1 2 and 10 mL of absolute ethanol. The resulting solution was then cooled to 0 °C with oxygen bubbling through it. After several minutes, the reaction solution was irradiated with a 1000 W sodium lamp for 15 min. NCS (40.0 mg, 1.1 eq.) was added, the reaction maintained at 0 °C for 60 min and then at room temperature for 3 h. The solvents were evaporated and the residue purified by preparative TLC, eluting with CH 2 C1 2 .
  • Example 15 Synthesis of Compound 14. 4- (Hexafluoroisopro ⁇ poxy) -4- (3-hydroxyphenyl)spiro[1,2-dioxetane-3 , 2 ' -tricyclo- [3.3.l.l 3 ' 7 ]decane] .
  • the vinyl sulfide of Example 2(b) (0.102 g) was irradiated for 10 min in 20 mL of CH 2 Cl 2 containing 2 mg of methylene blue with continuous oxygen bubbling at* -78 °C using a 1000 W Na lamp.
  • This oil was redis ⁇ olved in a small amount of CH 2 C1 2 and the product isolated by preparative TLC using 30% ethyl acetate in hexane yielding 8.1 mg of the product as a colorless oil. Reaction of a sample of the dioxetane with an excess of tetrabutylammonium fluoride in DMSO produced green chemiluminescence.
  • Example 16 Synthesis of Compound 15. 4-(3-Hydroxyphenyl) - 4- (2 ,2,2-trichloroethoxy) spiro[1, 2-dioxetane-3 , 2 ' -tricyclo- [3.3.l.l 3 ' 7 ]decane] .
  • the vinyl sulfide [ (3-hydroxyphenyl) - ethylthiomethylene]tricyclo[3.3.l.l 3 ' 7 ]decane (102 mg) was irradiated for 20 min in 10 mL of CH 2 C1 2 containing 1 mg of methylene blue with continuous oxygen bubbling at -40 °C using a 1000 W Na lamp.
  • N-Chlorosuccinimide (1 eq. ) was added. After 10 min, 3 mL of 2,2, 2-trichloroethanol was added and the solution warmed to room temperature. After an additional 30 min, TLC showed a new material which emitted blue-green light when the plate was heated. The solution was evaporated, redis ⁇ olved in a ⁇ mall amount of CH 3 OH and evaporated.
  • Example 17 Svnthe ⁇ is of Compound 16. 4- (2,2-Dichloro- ethoxy) -4- (3-hydroxyphenyl)spiro[1, 2-dioxetane-3, 2 ' - tricyclo- [3.3.l.l 3 - 7 ]decane] .
  • the vinyl sulfide [(3- hydroxyphenyl) ethylthiomethylene] tricyclo [3.3.l.l 3 ' 7 ] decane (111 mg) was irradiated for 16 min in 10 mL of CH 2 C1 2 containing 1 mg of methylene blue with continuous oxygen bubbling at -78 °C using a 1000 W Na lamp.
  • Chlorosuccinimide (50 mg, 1 eq. ) was added. After 10 min. 60.7 ⁇ L of 2,2-dichloroethanol (2 eq. ) was added and the solution warmed to room temperature. After an additional 30 min, TLC showed a new material which emitted blue-green light when the plate was heated. The solution was evaporated, redissolved in a small amount of CH 2 C1 2 and filtered.
  • Example 18 Synthesis of Compound 17. 4- (3-Hydroxyphenyl) - 4- (2,2, 3 ,3 , 3-pentafluoropropoxy) ⁇ piro[1, 2-dioxetane-3 ,2 ' - tricyclo[3.3.1. I 3 ' 7 ] decane] .
  • the vinyl ⁇ ulfide of Example 2(b) (0.112 g) was irradiated for 10 min in CH 2 C1 2 containing methylene blue with continuous oxygen bubbling at -78 °C using a 1000 W Na lamp. The solution wa ⁇ poured into a ⁇ olution of mercuric acetate (100 mg, 0.84 eq.
  • Example 19 Synthesis of Compound 18. 4- (2,2 , 3 , 3, 4, 4, 4- Heptafluorobutoxy) -4- (3-hydroxyphenyl) ⁇ piro[1, 2-dioxetane- 3,2 ' -tricyclo[3.3.l.l 3 - 7 ] decane] .
  • the vinyl ⁇ ulfide of Example 2(b) (0.100 g) wa ⁇ irradiated for 10 min in 20 mL of CH 2 C1 2 containing 2 mg of methylene blue with continuou ⁇ oxygen bubbling at -78 °C using a 1000 W Na lamp. The solution was poured into a solution of mercuric acetate
  • Example 20 Synthesis of Compound 19. 4- (2' , 6 ' -Difluoro- phenoxy) -4- (3-hydroxyphenyl) ⁇ piro[1, 2-dioxetane-3 ,2 ' - tricyclo[3.3.1. I 3 - 7 ]decane] .
  • the vinyl ⁇ ulfide of Example 2(b) (0.100 g) was irradiated for 10 min in 20 mL of CH 2 C1 2 containing 2 mg of methylene blue with continuous oxygen bubbling at -40 °C using a 1000 W Na lamp. NCS (44.7 mg, 1 eq. ) was added followed after 15 min by 100 mg (2.3 eq.
  • Example 21 Synthesis of Compound 20. 4-Methoxy-4- (3- pivaloyloxyphenyl)spiro[1,2-dioxetane-3,2 ' -tricyclo- [3.3.1.l 3 ' 7 ]decane] .
  • the vinyl ⁇ ulfide of Example 4(a) (0.051 g) wa ⁇ irradiated in 20 mL of CH 3 OH containing ca. 1 mg of Rose Bengal with continuous oxygen bubbling at -78 °C u ⁇ ing a 1000 W Na lamp.
  • iodine 0.096 g
  • Example 22 Synthesis of Compound 21. 4- (3-t-Butyldimethyl- silyloxyphenyl) -4-methoxyspiro[1, 2-dioxetane-3 , 2 ' -tricyclo- [3.3.1.I 3 - 7 ]decane] .
  • Thi ⁇ example demonstrates the inter- conversion of triggerable dioxetanes by replacing one protecting group with another. A 152 mg portion (1 mmol) of t-butyldimethylsilyl chloride was added to a solution of imidazole (69 mg, 1 mmol) in 2 mL of CH 2 C1 2 causing formation of a white precipitate.
  • Example 23 Synthesis of Compound 22. 4- (3-t-Butyldiphenyl- ⁇ ilyloxyphenyl) -4-methoxy ⁇ piro[1, 2-dioxetane-3, 2 ' -tricyclo- [3.3.l.l 3 - 7 ]decane] .
  • Thi ⁇ example further demon ⁇ trates the interconversion of triggerable dioxetanes.
  • a 950 mg portion (3.45 mmol) of t-butyldiphenylsilyl chloride was added to a solution of imidazole (236 mg, 3.47 mmol) in 25 mL of
  • Example 24 Synthesis of Compound 23. 4- (Methoxy) -4- (3-bis- (cyanoethy1)phosphoryloxyphenyl) ⁇ piro[1,2-dioxetane-3 ,2 ' - tricyclo[3.3.1.I 3 - 7 ]decane] .
  • Examples 24 and 25 further demonstrate the interconversion of triggerable dioxetanes.
  • a solution of anhydrous pyridine (3.0 mL, 37 mmol) in 10 mL of CH 2 C1 2 was placed under argon and cooled to 0 °C.
  • POCl 3 (1.608 g, 10.5 mmol) was added and the solution stirred for 15 min to cool.
  • the solid material was filtered off and washed with 50 mL of methanol.
  • the methanol washes and reaction solution were combined and evaporated under reduced pre ⁇ ure yielding a white ⁇ olid.
  • the solid was freed of impurities by again dis ⁇ olving in methanol, filtering and evaporating the methanol and cry ⁇ tallizing from methanol/acetone yielding 0.986 g of white ⁇ olid which wa ⁇ identical by X H NMR to an authentic sample (see U.S. Patent 5,004,565) .
  • Example 26 Alternate Synthesis of Vinvl Sulfide from Example Kh) .bv Ti-Mediated Coupling
  • (a) Synthesis of ethyl 3-methoxythiobenzoate.
  • m-anisoyl chloride 8.53 g
  • pyridine 4.85 mL
  • ethanethiol 3.73 g
  • the solution was diluted with 100 mL of methylene chloride, washed with saturated sodium bicarbonate then washed several times with water and dried.
  • Example 27 Comparison of Rates of Base-Induced Decay of Hvdroxvphenvl Alkoxv or Arvloxv Dioxetanes.
  • the half-live ⁇ of decay of chemilumine ⁇ cence (t 1/2 ) of hydroxy-dioxetane ⁇ correlate with the times required to reach the maximum light intensity (I max ) in the alkaline phosphata ⁇ e-triggered decompo ⁇ ition of the corre ⁇ ponding phosphate-dioxetanes under the same condition ⁇ .
  • the half-life of decay of lumine ⁇ cence of the hydroxy dioxetane i ⁇ therefore, u ⁇ eful for predicting the grow-in kinetics of light emission for pho ⁇ phatase triggering of phosphate dioxetanes; i.e.
  • a fast t 1/2 for the hydroxy dioxetane indicates that the corresponding phosphate dioxetane is expected to reach I max more quickly. It is expected that, for example, the phosphate derivatives of dioxetanes 14-19 as well as others which can be produced by the process described herein will be useful in alkaline phosphatase-linked as ⁇ ay ⁇ known in the art.
  • Example 28 Comparison of Rates of Base-Induced Decay of a Hydroxyphenyl Alkylthio Dioxetane.
  • Surfactant A is CTAB
  • B is poly(vinylbenzyltributylphosphonium chloride)
  • C i ⁇ poly(vinylbenzyltributylphosphonium chloride) -co-poly(vinylbenzyltrioctylphosphonium chloride)
  • D i ⁇ l-trioctylphosphoniummethyl-4-tributylphosphonium- methylbenzene dichloride.
  • Example 29 Fluoride Induced Chemiluminescence of Alkoxv and Aryloxy-Dioxetanes.
  • a portion of each of the purified dioxetanes 6-23 was separately mixed with a solution of 0.1 M tetrabutylammonium fluoride (TBAF) in DMSO causing a brief flash of blue-green light which could be ⁇ een in a darkened room by eye.
  • TBAF tetrabutylammonium fluoride
  • Chemilumine ⁇ cence persisted for several seconds. Light emission produced in this manner could also be produced with the dioxetane deposited on a silica gel TLC plate.
  • Example 30 Fluoride Induced Chemiluminescence from Sulfur- Substituted Dioxetanes. A portion of each of the dioxetanes 1-5 was separately mixed with a solution of 0.1 M TBAF in DMSO cau ⁇ ing a brief flash of yellow to reddish light which could be seen in a darkened room by eye. Chemiluminescence persisted for several seconds.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
  • Heterocyclic Compounds That Contain Two Or More Ring Oxygen Atoms (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne des sulfures de vinyle portant des substituants arylxoxy protégés et sur le procédé pour leur préparation. Dans le procédé, un sulfur de vinyle est formé par couplage d'un composé de cétone et d'un composé de thioester avec un réactif de titane à valeur faible pour former deux liaisons carbone-carbone dans la même étape. Les composés de sulfur de vinyle sont utiles comme intermédiaires qui sont convertibles en dioxétanes à substitution soufre déclenchables par addition de l'oxygène à la double liaison. Les dioxétanes à substitution soufre sont en outre utiles pour produire une chimioluminescence ou pour une conversion ultérieure en dioxétanes déclenchables à substitution alcoxy, alcényloxy, alcynyloxy, aryloxy, aralkyloxy ou acyloxy. Les dioxétanes déclenchables sont utiles pour produire une chimioluminescence et pour détecter des agents activants dans des dosages tels que les immunodosages et les dosages d'hybridation d'acide nucléique.
PCT/US1996/009249 1995-06-20 1996-06-19 Nouveaux composes de sulfure de vinyle et leur procede de preparation WO1997000849A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
DE69624374T DE69624374T2 (de) 1995-06-20 1996-06-19 Neue vinylsulfid-verbindungen und ein verfahren zu ihrer herstellung
AT96923225T ATE226188T1 (de) 1995-06-20 1996-06-19 Neue vinylsulfid-verbindungen und ein verfahren zu ihrer herstellung
CA002222788A CA2222788C (fr) 1995-06-20 1996-06-19 Nouveaux composes de sulfure de vinyle et leur procede de preparation
EP96923225A EP0869938B1 (fr) 1995-06-20 1996-06-19 Nouveaux composes de sulfure de vinyle et leur procede de preparation
AU63793/96A AU709184C (en) 1995-06-20 1996-06-19 Novel vinyl sulfide compounds and a process for their preparation
US08/981,448 US5883287A (en) 1995-06-20 1996-06-19 Vinyl sulfide compounds and a process for their preparation
JP9503860A JP3023993B2 (ja) 1995-06-20 1996-06-19 新規なスルフィド化合物及びその製法

Applications Claiming Priority (2)

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US49271595A 1995-06-20 1995-06-20
US08/492,715 1995-06-20

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Cited By (1)

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CN102030779A (zh) * 2010-11-17 2011-04-27 云南瑞亘生物科技有限公司 一种免疫分析用化学发光物amppd的制备方法

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US6528652B1 (en) 1999-01-21 2003-03-04 Chronimed Composition and device for detecting leukocytes in urine
US6348324B1 (en) 1999-01-21 2002-02-19 Hypoguard America Limited Composition and device for detecting leukocytes in urine
US7735278B2 (en) * 2007-03-29 2010-06-15 Stronggo Llc Edge-adapted detectable warning tiles with bottom-side extensions
US7779591B2 (en) * 2007-03-29 2010-08-24 Stronggo Llc Tiles with bottom-side extensions and method for installation

Citations (2)

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Publication number Priority date Publication date Assignee Title
US5068339A (en) * 1986-07-17 1991-11-26 Board Of Governors Of Wayne State University Enhanced chemiluminescence from 1,2-dioxetanes through energy transfer to tethered fluorescers
US5220005A (en) * 1986-07-24 1993-06-15 Tropix, Inc. Substituted adamantyl dioxetanes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2038092T1 (es) * 1990-09-07 1993-07-16 The Board Of Governors Of Wayne State University Un compuesto de dioxetano.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5068339A (en) * 1986-07-17 1991-11-26 Board Of Governors Of Wayne State University Enhanced chemiluminescence from 1,2-dioxetanes through energy transfer to tethered fluorescers
US5220005A (en) * 1986-07-24 1993-06-15 Tropix, Inc. Substituted adamantyl dioxetanes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102030779A (zh) * 2010-11-17 2011-04-27 云南瑞亘生物科技有限公司 一种免疫分析用化学发光物amppd的制备方法

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AU6379396A (en) 1997-01-22
AU709184B2 (en) 1999-08-26
DE69624374D1 (de) 2002-11-21
DE69624374T2 (de) 2003-07-10
EP0869938A1 (fr) 1998-10-14
JPH10511101A (ja) 1998-10-27
US5883287A (en) 1999-03-16
JP3023993B2 (ja) 2000-03-21
EP0869938A4 (fr) 1998-10-14
EP0869938B1 (fr) 2002-10-16

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